The therapeutic activities elicited by mesenchymal stem cells (MSC) are in part mediated through paracrine action by the release of extracellular vesicles (EV) and secreted proteins. Collectively, these MSC-derived factors, referred to as the secretome product (SP), are intrinsically therapeutic and represent an attractive alternative to cell-based therapies. Herein, we developed a lyopreservation protocol to extend the shelf-life of the MSC-SP without compromising the structural or functional integrity of the vesicular components. The SP isolated from normoxia- and anoxia-exposed MSC elicited protective effects in an in vitro model of oxidative injury and the bioactivity was retained in the lyophilized samples. Three separate formulations of MSC-SP were isolated by tangential flow filtration using sucrose, trehalose, and mannitol as lyoprotectant agents. The MSC-SPs were lyophilized using a manifold protocol and the structural and functional integrity were assessed. The trehalose formulation of SP exhibited the highest EV and protein recovery after manifold-based lyophilization. To facilitate development as a therapeutic, a shelf lyophilization protocol was developed which markedly enhanced the recovery of EV and proteins. In conclusion, lyophilization represents an efficient method to preserve the structural and functional integrity of the MSC-SP and can be used to develop an off-the-shelf therapeutic. Mesenchymal stem cells (MSC) are widely recognized for their therapeutic and regenerative activities. These cells have multipotent differentiation potential and can be harvested from various postnatal tissues, including bone marrow, adipose tissue, and umbilical cord blood ,2,3. MSCsecretome product (SP), retain many of the intrinsic therapeutic properties of their parental cells of origin [These MSC-derived EV and soluble proteins, collectively referred to as the f origin ,15. Mesef origin . The solf origin ,18. The f origin ,20. The f origin .The MSC-SP is an attractive alternative to MSC cell-based therapeutics for several reasons. The MSC-secretome has a better safety profile, due to the acellular nature of the material; namely, it is less immunogenic, and has negligible tumorigenic potential ,23. The Herein, we sought to extend the biological half-life of the MSC-SP towards the creation of a ready-to-use, off-the-shelf product. Using tangential flow filtration, three batches of MSC-SP, each diafiltrated with a different sugar-based formulation, were isolated and tested. A shelf-based lyophilization protocol was then developed to create a freeze-dried MSC-SP with an extended shelf-life, and the effects on structural properties and the biological activity were assessed after recovery.l-glutamine and 0.1% gentamicin . HepG2 hepatocarcinoma cells  were cultured on collagen-coated plates in Dulbecco’s modified eagle medium  supplemented with 10% FBS  and 1% penicillin-streptomycin.Primary human bone marrow-derived mesenchymal stem cells  were cultured in MSC basal media supplemented with 10% fetal bovine serum (FBS), 2% Five hundred milliliters of MSC was transferred to a 500 mL reservoir and loaded into the kR2i Kross Flow Tangential Flow Filtration (TFF) system furnished with a modified polyethylersulfane (mPES)-coated D02-E010-05-S hollow fiber filter . The TFF system was set to run on manual mode at a flow speed of 30 mL/min. The permeate was collected and subsequently passed through a sterile 0.22 µM filter and stored at 4 °C for future use.2/95% N2 for 24 h (aMSC)) or kept under normoxic conditions for an additional 24 h (nMSC). MSC-conditioned media (MSC-CM) were then collected and stored at 4 °C.Passage three MSC were cultured in vesicle-depleted complete media for 24 h. The MSC were then subjected to anoxia . The flow rate was maintained at 30 mL/min and the shear rate did not exceed 2000 s−1. The product was concentrated to reduce the volume to 5 mL, and then diafiltrated five times with either PBS, 5% w/v sucrose, 4% w/v mannitol or 4% w/v trehalose. The material was further concentrated two times to achieve a final volume of 3 mL. The isolated MSC-secretome products (MSC-SP) were stored at 4 °C. The filter was washed with PBS between consecutive isolations.The MSC-CM underwent sequential centrifugation at 300× −3 mBAR and −81 °C . The resulting lyophilized MSC-SP samples were reconstituted in 100 µL of the appropriate lyoprotectant agent. The samples were evaluated by BCA assay and NTA analysis to assess the protein and particle recovery, respectively.Following isolation by TFF, the resulting MSC-SP samples were separated into equal volume aliquots and one aliquot of each sample was stored at 4 °C and the others were stored overnight in a cool cell at −80 °C. The frozen MSC-SP samples were lyophilized for 48 h using a freeze FreeZone Plus 6, which was maintained at 3 × 10w/v trehalose (vehicle control), 8.00 × 106 particles of fresh or 1.00 × 108 particles of the lyophilized trehalose formulation of MSC-SP. After 24 h, the cells were incubated with 20 µM of DCFDA for 45 min in complete darkness at 37 °C. The media were then removed and replaced either with media or media supplemented with 0.5 mM tert-butyl hydrogen peroxide (TBHP) for 1 h. ROS specific production was quantitated by measuring the fluorescence intensity at an excitation wavelength of 485 nm and emission of 535 nm.ROS levels were detected using 2′,7′-Dichlorodihydrofluorescein acetate (DCFDA). This is a cell-permeable probe that is non-fluorescent. Once taken up by cells, DCFDA is converted by intracellular esterases and oxidized by intracellular ROS to 2′,7′dichlorofluorescein, which is highly fluorescent. The increase in fluorescence is a sensitive indicator of the presence of ROS. HepG2 cells were seeded onto a black collagen-coated 96-well plate at a concentration of 10,000 cells/well. After an overnight attachment period, the cells were treated with 4% A shelf-based lyophilization protocol using the VirTis AdVantage 2.0  was developed to improve the protein and EV recovery. In brief, fresh MSC-SP aliquot samples were subjected to thermal treatment in which the shelf temperature was gradually reduced to −40 °C for 150 min, while the pressure was maintained at 760 Torr. The pressure was subsequently reduced to 200 mTorr for 300 min and the shelf temperature was maintained at −40 °C. The primary drying phase was initiated by progressively increasing the shelf temperature to 20 °C over a 900-min period, while the pressure was kept at 200 mTorr . The resNanoparticle tracking analysis of the MSC-SP samples was performed using a Nanosight LM10 . Three videos were captured, and the average size and particle concentration were quantified. The NTA 3.3 software was used for data analysis.A BCA assay was performed to quantify the protein content of the samples according to the manufacturer’s protocol . Briefly, 80 µL of the reagent A + B solution was added to each well containing the MSC-SP samples. The plate was incubated at 37 °C for 30 min in complete darkness and the absorbance was subsequently measured at 562 nm using a FLUOstar omega plate reader . A standard curve of the blank corrected data was generated using a four-parameter fit.w/v) [w/v sucrose [w/v sucrose, 4% w/v trehalose and 4% w/v mannitol. Following the lyophilization of the MSC-SPs, the samples were reconstituted, and the EV and protein contents were quantified and compared to the respective fresh counterpart MSC-SP sample. The nanoparticle recovery in the PBS MSC-SP formulation exceeded 150%, which is suggestive of EV shearing  , as well sucrose . Therefoshearing A, whereashearing B. Therefp < 0.05, Previous research has shown that MSC are dynamic cells and alter their secretome profile to adapt to changing microenvironments . To devew/v trehalose and 4% w/v mannitol. The SPs were separated into equal-volume aliquots and one aliquot of each formulation was freeze-dried using the newly developed shelf-based protocol. The MSC-SPs preserved using the shelf-based protocol had markedly higher EV and protein recoveries compared to those prepared with the manifold-based lyophilization protocol (With the advent of new lyophilization technology, we developed and tested a shelf-based lyophilization protocol specifically designed for the preservation of cell-derived trophic factors, including EV and secreted proteins. As before, we prepared three formulations of MSC-SP using lyophilization excipients, 5% sucrose, 4% protocol A,B. The protocol A. Differprotocol .MSC-EV and SP represent attractive therapeutics for a variety of reasons. These materials offer lower immunogenicity profiles, are relatively inexpensive to isolate compared to the costs associated with maintaining MSC and can be modified to enhance the therapeutic effectiveness and/or to ensure targeted delivery ,31. For In the present study, we developed a method to generate an “off-the-shelf” MSC-derived acellular therapeutic to address some of the abovementioned issues. Herein, we utilized TFF to isolate the secretome product from bone marrow-derived MSC. We developed a manifold-based lyophilization protocol to prepare freeze-dried formulations of MSC-SP. The resulting lyophilized MSC-SP retained their bioactivity in vitro. Lastly, we developed an improved shelf-based lyophilization protocol that resulted in lyophilized MSC-SP with superior EV and protein recoveries, compared to those prepared using a manifold-based protocol. The MSC-SP lyophilized with the shelf protocol retained their structural integrity.Compared to the traditional method of EV isolation, ultracentrifugation, TFF enables the isolation of both EV and secreted proteins in a GMP-compliant manner, which can be scaled up for increased production. The ability to process high volumes of MSC-CM addresses one of the challenges in developing MSC-SP-based therapeutics for clinical use. Extracellular vesicles isolated by this method retain their structural integrity, since they are not subjected to the high speeds required for ultracentrifugation-based EV isolation . This feA challenge in translating MSC-EV and MSC-SP based therapeutics to the clinic is the optimization of storage conditions to minimize the loss of material and to retain functional activity. Previous studies have shown that storage at −80 °C resulted in a minimal reduction in EV count; however, increased storage periods resulted in the formation of aggregates . The EV The use of sugar-based excipients as protective agents has been explored during the preservation of artificial or biologically derived nanovesicles . DisacchIn this study, we assessed the effectiveness of three disaccharide excipients in two lyophilization protocols. With the manifold protocol, rapid temperature reductions could pose significant stressors to the EV and protein constituents. Following manifold freeze drying, the MSC-SP formulated with trehalose had the highest protein recovery and had an EV recovery that was similar to the other SP formulations. Compared to sucrose, trehalose may have a greater ability to disrupt ice crystal formation, and may be a more effective lyoprotectant . ConversWe have developed a method to prepare an off-the-shelf MSC-SP-based acellular therapeutic with an extended biological shelf life. TFF was used to isolate the protein and EV constituents from MSC culture supernatant in a scalable manner. Trehalose was the most effective at preserving the structural integrity of the MSC-SP following manifold-based lyophilization. This formulation of lyophilized MSC-SP also retained biological activity in vitro. A shelf protocol was developed to further improve the recovery of the freeze-dried MSC-SP constituents, with the highest recovery observed using mannitol. Thus, the appropriate selection of lyophilization protocols and protectants can be used to preserve the structural and functional integrity of MSC-SP, and thereby can address a major obstacle to developing MSC-based acellular therapeutics for clinical applications.


The findings show that more than half (53.7%) of school leaders had insufficient health literacy. Participants with insufficient health literacy scored significantly higher in the following factors: exhaustion related to work situation (p = 0.029), psychosomatic complaints (p < 0.001), attitude about vaccination  (p < 0.001), level of informing on COVID-19 related information  (p < 0.001), and level of confusion about COVID-19-related information  (p < 0.001). In a linear regression model predicting attitude about coronavirus vaccination, age  and health literacy  were the negative predictors, F = 11.859, p < 0.001. For the linear regression model adjusted for sex and age for predicting health literacy, the model was insignificant. Despite being a highly educated group, this study reveals that one in two Hong Kong school leaders have insufficient health literacy. Inadequate health literacy was strongly associated with a negative attitude about vaccination, low information, and confusion about COVID-19-related information. Additionally, insufficient health literacy was associated with the two secondary symptoms of burnouts. The study highlights an urgent need to develop intervention programmes to promote the COVID-19-specific as well as overall health literacy of the school leaders.To date, we know little about COVID-19-related health literacy among school leaders, particularly in East Asia. The present study aimed to assess the level of COVID-19-related health literacy and associated factors  among school leaders in Hong Kong. A cross-sectional study of 259 school leaders was carried out during the COVID-19 pandemic between April 2021 and February 2022. COVID-19-related health literacy using HLS-COVID-Q22, three subscales of self-endangering work behaviour scales , and two dimensions of Burnout Assessment Tool (BAT)  were used. The study employed independent sample  Health literacy is a key contributor to an individual’s health and is recognised as a central pillar of health promotion . AccordiInvesting in health literacy of children and adolescents’ promises benefits for their health and well-being development over time ,11. TherFew studies have examined health literacy among school personnel. A recent German study of 680 school leaders indicated that 29.3% had limited health literacy . With reDriven by the current COVID-19 pandemic, health literacy has been the subject of increasing attention in different occupational groups, whilst primarily focusing on healthcare workers  and teacEvidence shows that there is an association between low levels of health literacy and poorer health outcomes ,34. Low In terms of SARS-CoV-2 vaccination hesitancy, recent findings suggested that it was found to be associated with health literacy in the general population . A studyMaking informed health decisions might be helpful in occupational and health contexts, such as when addressing issues like recuperating from illness, missing work due to illness, and reintegrating back into society and the workforce . AccordiDespite mounting research examining the levels of health literacy, few researchers have investigated the health literacy levels of school leaders. To the best of our knowledge, no reports have examined the health literacy of school leaders in Hong Kong during the COVID-19 pandemic. Moreover, there are no reports addressing the associated factors in relation to the level of health literacy. Therefore, the aims of this study were to (a) assess the COVID-19-related health literacy among school leaders in Hong Kong, (b) examine the associated factors in affecting the health literacy of school leaders, (c) examine the differences in health literacy in relation to different demographic variables, (d) examine the differences in health literacy in relation to the level of COVID-related information received, and (e) examine the effect of health literacy on COVID-19 vaccine hesitancy among school leaders in Hong Kong.https://COVID-hl.eu, accessed on 17 September 2022), a global research network involving over 150 researchers from 75 countries focusing on health literacy research in relation to COVID-19. We used a descriptive cross-sectional design. As part of a larger study which aimed to examine the work and health situation of the school community in Hong Kong, this study was conducted between April 2021 and February 2022 during the period of strict social distancing measures implemented in Hong Kong in a zero-COVID policy context. The English-language survey was adapted from Dadaczynski et al. [This study is part of the COVID-19 Health Literacy Network  in primary, secondary and special schools in Hong Kong. First, we sent email invitations to the online survey in both English and Chinese to the principals of 1130 schools registered with the Education Bureau of the government in Hong Kong, including 561 primary schools, 477 secondary schools and 36 special schools. We also contacted our school networks by phone and mailed hardcopy questionnaires to 243 school principals after obtaining verbal consent. In addition, to expedite the participant recruitment, principals were recruited via authors’ personal networks conducted though social media platforms . We also recommended that respondents invite eligible participants to participate. The final sample consisted of 259 school leaders. According to the G Power software version 3.1.9.7 for Windows, the required sample size was calculated as 213, with an effect size of 0.1, type I error of 0.05, power of 0.95 and 18 potential predictors in multiple linear regressions. We used a self-reported 22-item corona-specific health literacy scale (HLS-COVID-Q22), with the Cronbach alpha value of 0.94, to assess participants’ ability to comprehend and apply health information related to COVID-19 ,56. PartThe exhaustion related to work situation was assessed using the “exhaustion” subscale of the Burnout Assessment Tool (BAT). Participants were asked to rate how often the statements applied to them on a five-point scale, from 1 (never) to 5 . The total exhaustion scores were calculated by the sum score of the 3 items and divided by the number of items. A higher score shows stronger exhaustion. The “Exhaustion” subscale has satisfactory reliability (Cronbach α = 0.85–0.87) . Self-reSelf-endangering work behaviour is a coping mechanism that buffers diminished well-being in response to heavy workloads and a lot of pressure to organise oneself . Three sWe measured how well informed participants feel about the coronavirus or the corona pandemic with a 5-point scale, ranging from 1 (very informed) to 5 (insufficiently informed) . The feeThe willingness to get vaccinated against COVID-19 if offered was also assessed by rating on a 6-point scale, ranging from 1  to 5 (certainly not) and 6 . A self-reported survey measuring attitudes towards vaccination was adopted. It consists of 5 items, including “vaccinations are important to protect me and my family.”. The item “Vaccination is in line with my religious beliefs” was separated into 2 items: “Vaccination is compatible with my attitudes”, and “Vaccination is compatible with my religious beliefs.” Participants were asked to rate how much they agree with the statements, by rating on a 4-point scale, ranging from 1  to 4 . The mean score of the items was calculated and used. The Cronbach alpha value of the present study was 0.882. To collect information about the demographic and work-related characteristics of the participants, closed questions were used, which revealed information regarding gender , age, school types , position at school .SD), whereas categorical variables with number (n) and percentage (%). Independent sample t-tests were used to compare the difference in health literacy among groups, such as gender, positions at school and type of schools, while associated factors of COVID-19-related health literacy were tested using bivariate and multivariate linear regression models, including the level of informing on COVID-related information, level of confusion on COVID-related information, self-endangering work behaviour , work satisfaction and secondary symptoms of burnout . Health literacy was used as a dependent variable for the bivariate analysis to detect differences with regard to demographic and work characteristics, but as an independent variable as a predictor for health outcomes and vaccine attitudes and behaviour. Separate multivariate linear regression models were used to investigate the effect of health literacy on vaccine attitude and perceived general health, respectively. Multicollinearity was ruled out in the analyses. For the multiple regression model developed, the F-test was used to validate the significance of the model. A p-value of less than 0.05 is considered statistically significant. Data analysis was undertaken using SPSS Version 27.0. Continuous variables were described using mean (M) and standard deviation (The sample was composed of 259 responses from school leaders . The meaSD = 0.42). A total of 46.3% of the participants was found to have sufficient health literacy, 47.5% had problematic health literacy and 6.2% showed inadequate health literacy. For individuals classified with sufficient health literacy, the mean health literacy score was 3.42 (SD = 0.31), whereas individuals with problematic or inadequate health literacy presented with a mean score of 2.88 (SD = 0.14) and 2.21 (SD = 0.36), respectively. To minimise the uneven effect of sample sizes, the present study has further classified health literacy level into dichotomised groups: sufficient health literacy (n = 138) and insufficient health literacy  (n = 121) for further analyses. The findings showed that health literacy varied between subgroups according to how well informed participants were of the COVID-19-related information and how confused they were about COVID-19-related information within the sample population. Participants who were well or very well informed on COVID-19-related information had higher health literacy levels (3.19 ± 0.46) compared to participants who were insufficiently, poorly or acceptably informed on COVID-19-related information (3.00 ± 0.37), t(255) = 3.528, p < 0.001. Participants who felt not at all or a little confused about COVID-19-related information had higher health literacy levels (3.12 ± 0.40) compared to participants who felt quite confused or very confused about COVID-19-related information (2.90 ± 0.54), t(255) = 2.73, p = 0.002. t-tests were used (SD = 0.80) scored significantly higher in “exhaustion related to work situation” of the BAT than those with sufficient health literacy , t(255) = 0.493, p = 0.029. For another dimension of BAT, principals with insufficient health literacy level  scored significantly higher in “psychosomatic complaints” than those with sufficient health literacy , t(253) = 3.56, p < 0.001. Regarding attitude towards vaccination, participants with insufficient health literacy level  scored significantly higher in attitude about vaccination  than those with sufficient health literacy , t(242) = 6.08, p < 0.001.To compare the difference in the scores among work-related factors  between each pair of groups, secondary symptoms of work-related burnout) and attitude about vaccination between the dichotomised groups, independent ere used . RespondSD = 0.68) scored higher than those with sufficient health literacy  in level of information about COVID-19-related information , t(255) = 4.467, p < 0.001. Participants with insufficient health literacy  scored higher than those with sufficient health literacy  in level of confusion about COVID-19-related information , t(252) = 3.964, p < 0.001. In addition, participants with insufficient health literacy  = 1.903, p = 0.07, with an R2 of 0.058.p = 0.002) and lower level of COVID-19-related health literacy ; F = 11.859, p < 0.001 with an R2 of 0.217. When the multiple linear regression model was adjusted for age and sex to predict attitudes towards the vaccine , a higheThe present study aimed to investigate Hong Kong school leaders’ COVID-19-related health literacy, their working conditions and aspects of health information, all within the context of the COVID-19 pandemic. To the best of our knowledge, this is the first health literacy study to explore COVID-19-related health literacy in school leaders in Hong Kong. We found that more than half (53.7%) of the school leaders in Hong Kong had insufficient health literacy, including 47.5% problematic health literacy and 6.2% inadequate health literacy, and only 46.3% had sufficient health literacy. The mean health literacy score of this study (3.09 ± 0.42) was comparable to a recent study in Taiwan using the same tool (3.2 ± 0.4 ). ComparIn this study, the results showed no significant differences in health literacy levels between male and female participants. This finding is inconsistent with previous studies. Male school leaders in Germany had a higher percentage of limited health literacy than their female counterparts . In TaiwOur results showed that participants with sufficient health literacy would have a higher level of understanding of COVID-19-related information compared with those who had insufficient health literacy. This finding was consistent with a previous study conducted in Germany, where inadequate and problematic health literacy levels were more prevalent in people who reported themselves as “not so well informed” or “not well informed at all” . COVID-1In our study, results showed that higher scores in COVID-19-related health literacy was associated with a more positive attitude about vaccination. This finding aligns with research from Taiwan which shows the strong association of higher coronavirus-related health literacy and lower vaccine hesitancy , and alsIn terms of age, in line with the findings of a local study by Luk et al. , older aNo significant difference was found in the frequencies of performing self-endangering work behaviour, including work intensification, work extensification and quality reduction. According to previous literature, self-endangering work behaviour can be seen as a coping reaction in response to high work demands  and is uThe research presented has several limitations that should be addressed through future studies. First, our data were based on a self-report questionnaire by school leaders, and therefore may be subject to common method bias . Second,Despite being a highly educated group, one in two Hong Kong school leaders have insufficient health literacy. Our study fills the gap of health literacy literature of school leaders in East Asia, especially in China during the COVID-19 pandemic. The study highlights an urgent need to develop health educational programmes to promote and advocate the COVID-19-specific as well as overall health literacy of school leaders. The study also shows the need for organisational policies and strategies  focused


Nonetheless, further studies are needed to explore the pharmacokinetics of remdesivir and its relationship to clinical efficacy, and the present model may serve as a useful starting point for additional evaluations.Remdesivir is a direct-acting anti-viral agent. It was originally evaluated against filoviruses. However, during the COVID-19 pandemic, it was investigated due to its anti-viral activities against (SARS-CoV-2) virus. Therefore remdesivir received conditional approval for treatment of patients with severe coronavirus disease. Yet, its pharmacokinetic properties are inadequately understood. This report describes the population pharmacokinetics of remdesivir and its two plasma-detectable metabolites (GS-704277 and GS-441524) in healthy volunteers. The data was extracted from published phase I single escalating and multiple i.v remdesivir dose studies conducted by the manufacturer. The model was developed by standard methods using non-linear mixed effect modeling. Also, a series of simulations were carried out to test suggested clinical doses. The model describes the distribution of remdesivir and each of its metabolites by respective two compartments with sequential metabolism between moieties, and elimination from central compartments. As individual data were not available, only inter-cohort variability could be assessed. The estimated point estimates for central  volumes of distribution for remdesivir, GS-704277, and GS-441524 were 4.89 L (46.5 L), 96.4 L (8.64 L), and 26.2 L (66.2 L), respectively. The estimated elimination clearances of remdesivir, GS704277, and GS-441524 reached 18.1 L/h, 36.9 L/h, and 4.74 L/h, respectively. The developed model described the data well. Simulations of clinically approved doses showed that GS-441524 concentrations in plasma exceeded the reported ECThe online version contains supplementary material available at 10.1007/s00210-022-02292-6. Current management of patients with severe COVID-19 mainly focuses on immune response modulation and symptomatic therapy. In critically ill patients, management would also include supplemental oxygen and mechanical ventilation, along with the suppression of inadequate immune response. Healthcare providers also attempt to control secondary infections and thrombosis by either prophylaxis or treatment   parameters were estimated by standard methods using non-linear mixed effect modeling software , the volume of distribution for central and peripheral compartments (Vdc and Vdp), inter-compartmental clearance (Q), and formation clearance of metabolites (CLm). Terminal elimination half-life using regression (t½ cc) was also calculated using GraphPad Prism 8 . Microsoft Excel  and RStudio  were used for dataset construction, analysis, and graph generation.Estimated PK parameters included total body clearance . To incorporate this sudden increase of remdesivir concentrations into the model, 4% of the total administered dose was subtracted from the continuous infusion dose and given instantaneously at the end of the infusion. The choice of this method and the selected percentage were based on other published methodology .The most suitable model was developed using ordinary differential equations (ODE) in non-linear mixed-effect modeling software Table . The proθi is the estimated parameter for the mean concentration of the ith dose, θpopulation is the mean across doses, and ηi is a random effect describing the deviation of the PK parameter for the ith dose level from the typical PK parameter estimated for all doses. Parameter ηi is assumed to follow a normal distribution with a mean of zero and a variance of ω2.As no individual values were available, the obtained variability of fixed parameters reflects inter-cohort variabilities rather than inter-individual variability. And they lump the variability due to differences between volunteers in the different cohorts and they were defined with a log-normal distribution as.Cij is the observed value for the mean concentration of the dose i at time point j. Yij is the predicted concentration value of the dose i at time point j estimated by the model. Parameters a and b are additive and proportional residual errors, respectively.Additive, proportional, and combined error models were tested:Simulations were run using R  of remdesivir, GS‐704277, and GS‐441524 were 13.7 µM (2.39), 807 nM (173), and 726 nM (240), respectively, following the initial loading dose. GS‐704277 tmax was reached immediately after the end of the infusion, while GS‐441524 reached its tmax 1 h after the end of the infusion. The fraction of censored observations for remdesivir reached the value of 1 after 20 h following the initial 200-mg infusion, and 17 h for each sequential 100-mg infusion. In contrast, GS‐774277 and GS‐441524 did not reach the values of their reported LLOQ during the whole duration of therapy  and the half-life reached 29.36 h both estimated by simulation.Subsequent The simulation used the previously generated population parameters of fixed effects, the standard deviation of the random effects, and error model estimates .In this report, we were able to develop a compartmental non-linear mixed effect model that can describe the mean concentration vs. time course of remdesivir and its two detectable metabolites reasonably well, while also adapting for and describing inter-dose variability.Cmax) compared to other published non-compartmental analyses of remdesivir, GS‐441524, and GS‐704277  in SARS-CoV primary human airway epithelial cells Below is the link to the electronic supplementary material.


Cladocopium (C-type) is generally dominant in corals, Durusdinium (D-type) is found in more heat-resistant corals. Thus, the presence of D-type symbiont likely increases coral heat tolerance, and this symbiotic relationship potentially provides a hint to increase the stress tolerance of coral–algal symbioses. In this study, transcriptome profiles of Cladocopium- and Durusdinium-harboring Acropora solitaryensis  and algal photosystem functioning (Fv/Fm) under bleaching conditions (high temperature and light stress) were compared. Stress treatment caused algal photoinhibition that the Fv/Fm value of Symbiodiniaceae was immediately reduced. The transcriptome analysis of corals revealed that genes involved in the following processes were detected: endoplasmic reticulum (ER) stress, mitophagy, apoptosis, endocytosis, metabolic processes , and the PI3K-AKT pathway were upregulated, while DNA replication and the calcium signaling pathway were downregulated in both C- and D-corals. These results suggest that unrepaired DNA and protein damages were accumulated in corals under high temperature and light stress. Additionally, some differentially expressed genes (DEGs) were specific to C- or D-corals, which includes genes involved in transient receptor potential (TRP) channels and vitamin B metabolic processes. Algal transcriptome analysis showed the increased expression of gene encoding photosystem and molecular chaperone especially in D-type symbiont. The transcriptome data imply a possible difference in the stress reactions on C-type and D-type symbionts. The results reveal the basic process of coral heat/light stress response and symbiont-type-specific coral transcriptional responses, which provides a perspective on the mechanisms that cause differences in coral stress tolerance.Algal symbionts of corals can influence host stress resistance; for example, in the Pacific Ocean, whereas  Bleaching phenomena cause fatal impact in the most cases to host corals because the loss of alga does not provide the expected amount of energy source to the host , heat-shock, and photosystem-constitutional proteins have been reported as the heat stress-responsive transcripts   to a stress-resistant type, Durusdinium   Baker, . The inf) Baker,  and by t) Baker, . D-type ) Baker, . The C-t) Baker, . The eff) Baker, . Thus, t) Baker, .Fv/Fm) of symbiont was measured by pulse amplitude-modulated (PAM) fluorometry and performed large-scale gene expression analysis of coral–algal associations.However, there are knowledge gaps in the difference in bleaching sensitivity derived from each symbiont due to the difficulty of preparing corals associated with symbionts, although there are a few molecular studies recently reported  and CCMP 2466  were purchased from the Bigelow Laboratory for Ocean Sciences . Acropora solitaryensis larvae were generated from ~5 different colonies located in the area of 32°48′16.5″ N 132°39′11.1″ E  as described by Omori and Iwao  and the water temperature was increased gradually from 27 to 31°C over 21 h. The temperature was controlled by a thermo-controller . We confirmed that the temperature accuracy was within ± 0.5°C by thermometer under digital microscope . This bleaching condition was determined in the preliminary experiments conducted with juvenile A. solitaryensis corals. Coral polyps were photographed with a stereomicroscope  mounted on a CMOS camera . These photographs enabled us to confirm the symbiont cell densities in the coral tissue, and to visualize the decrease therein  treatment was also used as the control for the first day of stress (stress day 1) treatment. Here, it was assumed that the expression pattern would not change even if the fixation time for RNA-seq was shifted by 1 day, since the condition is stable after 2 months of culture. Corals were fixed on day 1  and on day 2 (after 30 h at 31°C) in RNAlater  and stored at −80°C. The control population was maintained at 27°C and 80 μmol m−2 s−1 (12-h light–dark cycle) and fixed after 30 h.Second, other corals were exposed to the same stress conditions described above and fixed after 6 (29°C) or 30 h (31°C) to prepare the samples for transcriptome analysis. Approximately 90 colonies each were prepared for C- and D-type corals as the samples for RNA-seq. Among them, 30 corals each were used for the control, stress day 1 and stress day 2 treatments. These 30 corals were divided into two containers and prepared as the two biological replicates. For the control condition for RNA-seq analysis, corals were maintained at 27°C and 80 μmol mFv/Fm) of endosymbiotic algae was monitored by PAM fluorometry  .The photosynthetic parameter (2), the surface area of photographed corals was calculated using ImageJ software. These corals do not differ in height; thus, only the surface area was estimated. To clarify that a bleaching response was occurring, algal cell density ratios of “stress day 2” / “control” were calculated.After the end of the stress experiment, corals were fixed in 3% formaldehyde to count symbiont cells inside corals . The fixed corals were decalcified in solution containing 0.5 M EDTA and homogenized in 0.01% Triton X using the method described by Yuyama and Higuchi . Then, sPoly(A) RNA isolation from total RNA was performed with a protocol of Next Poly(A) mRNA Magnetic Isolation Module . The RNA quality of each sample was checked using an Agilent 2100 Bioanalyzer  before producing the libraries. The sequencing libraries were generated from poly(A) mRNA using the NEBNext Ultra RNA Library Prep Kit (New England Biolabs). All libraries were sequenced on the HiSeq 4000 platform  by Macrogen Japan. The resulting 101-bp paired-end reads were first pre-processed  using the DNA Data Bank of Japan (DDBJ) Read Annotation Pipeline  Read Annotation Pipeline   by BLASTx search with an e-value cutoff of 1e-4. Gene Ontology (GO) enrichment analyses were performed on the annotated dataset of DEGs using the Database for Annotation, Visualization, and Integrated Discovery . In the analysis, annotated DEGs were compared with the annotation of the whole transcriptome, and GO terms enriched among the DEGs were identified. To summarize the major pathways involved in corals exposed to high-temperature/light, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses  was used . The Symbiodiniaceae database included the genomes of Symbiodinium minutum  and A. tenuis  very-sensitive algorithm. Then, the quantification of transcript abundances and identification of DEGs were performed by the same method used for the identification of coral transcripts. The eXpress (v. 1.5.1) program was used to quantify transcript abundances and estimate fragments per kilobase of transcript per million mapped reads (FPKM) values   of the C- and D-types were 0.582 ± 0.002 and 0.57 ± 0.013 (average ± standard error [SE]), at the beginning of the experiment; these values decreased to 0.042 ± 0.009 and 0.099 ± 0.002 (average ± SE), in the first 3 h. After this point, the photosynthetic activity of algae remained low until the end of the experiment. At 3 h, when the temperature was increased to 28°C under strong light, the D-type tended to show more photosynthetic activity than did the C-type. In the control treatment , no significant fluctuation of Fv/Fm was observed in either type; the Fv/Fm values were ~0.6. Fv/Fm values were significantly decreased under stress compared with control conditions, in both C- and D-types (p < 0.05), although no significant difference was observed between the two types. At the end of the experiment, the corals were fixed and decalcified to measure the symbiont densities. Cell density tended to decrease during the experimental period, with a 51% decrease in C-type and a 28% decrease in D-type compared to control conditions  remained largely unchanged until 6 h, and the color of the body gradually became white over the following 24 h . To invenditions .de novo assembly using reads from all samples, 663,795 contigs were generated. Subsequently, the protein-coding regions were estimated, and redundant sequences were removed, which resulted in 168,491 sequences. Among them, 40,036 contigs (DDBJ/ENA/GenBank accession codes ICPH01000001-ICPH01040036) assigned to the coral database by BLASTN search were used as a reference gene set of corals for subsequent analyses  and 30 h  after the start of the experiment. We sequenced cDNA libraries derived from each sample, and ~40 million reads were obtained from each. Sequence data were deposited in the DDBJ Read Archive (DRA008078). As a result of the analyses . All of analyses .p-values were selected and shown in via recombination  were mainly used for KEGG pathway analysis. De novo assembly of C- and D-type symbionts was performed, respectively, using FASTQ data of C-and D-corals, which resulted in the identification of 44,446 contigs (DDBJ/ENA/GenBank accession codes ICPI01000001–ICPI01003338) from C-type and 20,368 contigs (DDBJ/ENA/GenBank accession codes ICPJ01000001–ICPJ01004504) from D-type  are shown in m D-type . FASTQ rm D-type . Of themFv/Fm) of both Symbiodiniaceae immediately decreased, and the D-type maintaining a slightly higher Fv/Fm than the C-type under stress conditions. This is similar to the reports that the degree of Fv/Fm decline in the D-type was smaller than in the C-type under strong light conditions , and their stress response patterns and transcriptomic changes were investigated. By the stress treatment, a decrease in symbiotic cell density, which is a typical coral bleaching phenomenon, was observed. Photosynthetic activity  under high temperature conditions , Cladocopium assembly (accession codes ICPI01000001–ICPI01003338), and Durusdinium assembly .The raw fastq files for the RNA-seq libraries were deposited in the DDBJ Sequence Read Archive (DRA) under the accession number IY: study conception, design, data collection, and draft manuscript preparation. TH: statistical analysis, drafting, and writing. HT and TM: sample preparation. KI: drafting and writing. All authors contributed to the article and approved the submitted version.This work was supported by research grants from the Japan Society for the Promotion of Science  and the Environment Research and Technology Development Fund (No. 4–1806) from the Ministry of the Environment, Japan.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Coronavirus Disease (COVID-19) is caused by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). The SARS-CoV-2 virus is evolving continuously. The omicron variant of SARS-CoV-2 has the highest mutation in its spike protein, thus making the presently available vaccine ineffective or reducing its efficiency. Furthermore, the majority of the vaccines are constructed using a spike protein sequence from wild-type SARS-CoV-2. This raises the possibility of the virus evolving to the point where the vaccine's effectiveness is completely lost, even after booster doses. The study aims to develop a predictive vaccine as well as the epitopes for the updating of the vaccine sequences of currently available vaccines. In this study, following the immunoinformatics approach, predictive vaccine construction was done with the help of epitopes present on spike proteins of wild-type, delta, and omicron variants that encompass the majority of variants and possible new variants that arise from the combination of circulating variants.z score of − 1.4, − 1.7, − 1.4, − 1.7, and − 1.4, respectively, with a cluster size of 121 highest for the ACE2 receptor and 46 lowest for B-cell receptor chain A. The C-ImmSim simulation results indicate that the vaccine is generating both humoral and cell-mediated responses at a sufficient level throughout the month upon injection of the vaccine as an antigen.The vaccine that was constructed was stable and immunogenic. The vaccine was constructed with the help of 18 B-cell epitopes, 5 MHC class I epitopes, and 6 MHC class II epitopes. The epitope conservancy analysis suggests that the vaccine will work for the previously known variant of concern. The vaccine bound to TLR4, TLR2, B-cell receptor chains A and B, and ACE2 receptors with a The study's findings indicate that the vaccine was both stable and immunogenic, providing a sufficient level of immunity. Following experimental validation, the vaccine can be used, and the epitopes can be employed for therapeutic purposes such as antibody synthesis.The online version contains supplementary material available at 10.1186/s43088-023-00341-4. A novel coronavirus was identified in Wuhan, China, in December 2019; it is termed Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) [The previously known variant of concern alpha B.1.1.7) with mutation N501Y in the Receptor Binding Domain (RBD) of spike protein improved the affinity towards human the Angiotensin Converting Enzyme 2 (ACE2) receptor. Alpha also has the P681H mutation that affects the infectivity and replication of the virus, as well as mutations in the N-Terminal Domain (NTD) D178H, HV69-70del, and Y144del that affect the neutralisation by NTD-directed antibodies as they result in a conformational change in the NTD of spike protein . E484K i with mutThere were vaccine efficiency data and shreds of evidence of reduced neutralisation of some SARS-CoV-2 variants by post-vaccination sera. However, a deep understanding of correlates of protection was required to evaluate how this may impact vaccine effectiveness. Nonetheless, a possible update of the vaccine sequence is required for better immunity from the existing as well as emerging variants  showed tIn this study, a predictive multi-epitope vaccine was designed from the spike protein sequence of wild-type, delta, and omicron variants to cover a wide range of known and/or emerging variants. The multi-epitope vaccine has various epitopes to form the vaccine, giving it the advantage to work on multiple targets. These epitopes are the key factor that can help to update the vaccine sequence by providing data like antigenicity, population coverage, and conservation of these peptides in the spike protein sequences. This study focuses on spike protein and its sequences as they were the primary factor responsible for the interaction with the host (human) as well as for the construction of the vaccines. In addition, the emergence of new variants was facilitated by the mutations of spike protein sequences.https://www.ncbi.nlm.nih.gov/labs/virus/vssi/#/sars-cov-2). NCBI Virus is a centralised repository for viral sequence data, which includes RefSeq, GenBank, and other NCBI repositories. There is also a SARS-CoV-2 data hub. It was used to obtain the spike protein sequences of SARS-CoV-2 wild-type, delta, and omicron variants.The spike glycoprotein sequences were obtained from NCBI Virus (https://webs.iiitd.edu.in/raghava/abcpred/ABC_submission.html), which is an Artificial Neural Network (ANN)-based B-cell epitope prediction server. It predicts B-cell epitope with an accuracy of 65.93% in an antigen sequence based on a partial recurrent neural network in a fixed length pattern of 10, 12, 14, 16, 18, and 20 [For the prediction of the linear B-cell epitopes, the ABCpred server was used  of the Immune Epitope DataBase (IEDB) was used. TepiTool is a 6-step wizard that provides several top MHC binding prediction algorithms with hundreds of alleles for different species and, as a wizard, it provides a user-friendly interface with optimal cutoffs and guidelines [The T-cell epitopesare classified as cytotoxic T lymphocytes epitopes (MHC class I) and helper T lymphocytes epitopes (MHC class II) and are used to induce specific immune responses which are based on CD4idelines . For theA set of 12 MHC class I super-types was chosen for MHC class I binding epitope prediction, and the peptide length was set to 9 mers. The prediction method used was consensus, and the predicted peptide was chosen based on the predicted percentile rank with a cutoff value ≤ 1. Following that, the same procedure was repeated with a change in the predicted peptide method as the IC50 value with a cutoff of ≤ 500 nM. For MHC class II binding epitopes, the peptide length was fixed at 15 mers, the prediction method was IEDB recommended, and the predicted peptide was selected using the 7-allele method [v (http://www.ddg-pharmfac.net/vaxijen/VaxiJen/VaxiJen.html) a server to calculate antigenicity, has an accuracy of 70–89% [https://webs.iiitd.edu.in/raghava/algpred2/batch.html) is a server to identify allergen and non-allergen peptides. It was based on a machine learning algorithm to predict allergenic and non-allergenic proteins [https://webs.iiitd.edu.in/raghava/toxinpred/multi_submit.php) is a server for the prediction and designing of toxic and non-toxic peptides. It has 1805 toxic peptides with less than or equal to 35 residues [Epitope screening is the process of filtering previously procured epitopes to identify felicitous epitopes. In this elimination of duplicates, the removal of allergen and toxin peptides was done, while the most antigenic epitopes were preferred. This procedure is followed for both B-cell and T-cell epitopes. The Vaxijen server was used for antigenicity propensity analysis of epitopes. Vaxijen 2.0f 70–89% . The thrproteins . The thrresidues . The thrIn addition to antigenicity, non-allergenicity, and non-toxicity, the T-cell epitopes require additional properties to be a suitable candidate, i.e. immunogenicity and interferon-gamma induction (INF-γ) to be used for vaccine construction. The IEDB Class I immunogenicity tool was used for immunogenicity analysis to predict MHC I immunogenicity . The immhttp://tools.iedb.org/conservancy/) computes the extent of epitopes conserved within the protein sequence at an identity level set by the user [The IEDB's Epitope conservancy analysis tool was used to determine epitope conservancy. IEDB's epitope conservancy analysis (the user . This cohttp://tools.iedb.org/population/) was used [The T-cell peptide epitopes were used to estimate the patient population targeted by a vaccine based on these epitopes. For this purpose, the population coverage tool of the IEDB  and adjuvant . The linkers were used to better represent the epitopes as well as improve the immunogenicity and stability of a protein. Adjuvants help in improving immunogenicity and stability. Moreover, beta-defensin has a link to the activation of pathogen-specific innate and adaptive immunity, specifically beta-defensin 2 for enhancing type I immune response , 21. FurThe Basic Local Alignment Search Tool (BLAST) assessment of the vaccine construct against the human proteome was done to find any similar or homologous proteins to avoid any disruption in the human system.https://web.expasy.org/protparam/) of ExPASy was used to find the physical and chemical properties of the vaccine construct. ProtParam allows the computation of physical and chemical properties of protein stored in SwissProt/TrEMBL or user-provided protein sequences. Furthermore, the Scratch protein predictor was used for (http://scratch.proteomics.ics.uci.edu) SOLpro, DLpro, and ANTIGENpro for solubility upon overexpression, Disulphide bonds, and antigenicity.For the assessment of the vaccine construct, the ProtParam tool (https://npsa-prabi.ibcp.fr/cgi-bin/npsa_automat.pl?page=/NPSA/npsa_sopma.html) and PSIPRED tools were used with default parameters [For secondary structure prediction, the Self-optimized prediction method with alignment (SOPMA)  server, and predicted protein structure was validated with the help of the Ramachandran plot –28. The https://wenmr.science.uu.nl/haddock2.4/) was used [https://pipe.rcc.fsu.edu/meta-ppisp.html) [To test vaccine construct binding affinity with immune receptors, docking analysis was performed with the ACE2 receptor (PDB ID: 3SCI), TLR2 (PDB ID: 2Z7X), TLR4 (PDB ID: 3FXI), and BCR (PDB ID: 3KG5). To verify the binding affinity of vaccine constructs with class I and class II HLAs, the MHC class I HLA A*0201 (PDB ID: 4U6Y), HLA B*5101 (PDB ID: 4MJI), and MHC class II HLA DRB1*1402 (PDB ID: 6ATF) were docked. For docking analysis, the High Ambiguity Driven protein–protein DOCKing (HADDOCK 2.4) server  .https://kraken.iac.rm.cnr.it/C-IMMSIM/) was used to analyse the immune response generated by a vaccine injection by simulating the immune system [The C-ImtmSim server  at 18  of − 0.137 shows it as hydrophilic. The vaccine construct's aliphatic index of 83.88 indicates that it is a highly thermostable protein. The results of SOLpro, DLpro, and ANTIGENpro results for solubility upon overexpression, number of disulphide bonds, and antigenicity were 0.850797, 6, and 0.383958, respectively.The SOPMA result shows that the construct contains 45.10% alpha helices, 16.45% extended strands, 4.99% beta turns, and 33.46% random coils. Figure The tertiary structure of the vaccine was chosen from the best 5 models; the structure chosen has a C-score of − 1.8 and an estimated RMSD value of 11.8 ± 4.5 Å. The model was validated through a Ramachandran plot. The amino acids are depicted as highly preferred observations, which are depicted in green with a cross symbol, and a total of 432 amino acids are located in the highly favourable regions, which account for 80.148% of the total. The preferred observations are shown in brown with a triangle symbol; a total of 81 amino acids are present in this favourable region, accounting for 15.028%, while 26 amino acids are shown in a red colour circle, accounting for 4.824%. To reduce these questionable observations, the tertiary structure is refined by the ModRefiner server and further meliorated by the GalaxyRefiner server. In this tertiary structure refined by ModRefiner, questionable observations are reduced to 7 (1.299%), highly favourable observations are increased to 498 (92.393%), and favourable observations are 34 (6.308%) indicating that after structure refinement most of the amino acids are falling in the highly favourable region and favourable as well as questionable observations are reduced by more than 2 times with compare to the raw tertiary structure modelled by I-TASSER. The GalaxyRefiner server was used to further improve the structure. The GalaxyRefiner server returns five refined models; the best model was selected. In this tertiary structure refined by the Galaxyrefiner, questionable observations are 9 (1.67%), highly favourable observations are increased to 507 (94.063%), and favourable observations are 23 (4.267%)  in 2018 and 2020, in this case it performed poorly. The rank-1 model generated has a pLDDT score of 48.80, less than 50, and a pTM score of 0.2790. I-TASSER's best model has a TM score of 0.50 ± 0.15, a C-score of − 1.81, and an RMSD value of 11.8 ± 4.5 Å. The multiple sequence alignment (MSA) coverage, the Local Distance Difference Test (LDDT) per position and alignment error are in Additional file Since the SARS-CoV-2 spike protein interacts with the ACE2 receptor, SARS-CoV-2 contact and transmission can be regulated by ACE2 receptor blockers. For docking of a vaccine with the ACE2 receptor, HADDOCK server clustered 173 structures into 6 clusters, which represent 86% of the water-refined models that the HADDOCK server generated. The best model has a z score of − 1.4 and a HADDOCK score of − 79.4 ± 2.6. For docking with TLR4, HADDOCK server clustered 199 structures into 3 clusters, which represent 99% of the water-refined models. Likewise, for TLR2, 168 structures in 7 clusters represent 84% of water refined models; for B-cell receptor chain A, 151 structures in 9 clusters represent 75% of water refined models; for B-cell receptor chain A, 164 structures in 8 clusters represent 82% of the water-refined models. Similarly, for HLA in which the lowest percentage of the water-refined model was 48% for HLA-B chain A and the highest was 81% for HLA-D chain A. Table After 5 days of administration, the immune response generated by the vaccine as an antigen shows a sharp increase in IgM and IgG antibodies, as well as IgG isotypes that help in neutralising the SARS-CoV-2 by binding with specific epitopes. In terms of cell activation, B cells exhibit high presenting activity for the first 5 days and a high level of activation within 0–10 days before reaching saturation, whereas TH and TC cells are activated exponentially within 5 days. It also stimulated memory cells. The cytokines released after administration show a high level of interferon-γ, with interleukin 2 coming in second. The disparities in both levels were significant. A comparison was made between the predictive vaccine and the currently available vaccine based upon the C-ImmSim simulation results by utilising the S-2P protein sequence shows the predictive vaccine response to be parallel with available vaccine. It showed better result in B cell response , as well as the emergences of new variants contributed by the mutations of the spike protein sequences. The previous vaccines for SARS-CoV were mostly based on RBD of spike protein, while in this study whole spike protein sequences were considered instead of domains. In this study, the number of B-cell epitopes obtained after screening was high, indicating favourable interactions between B-cells and the virus. Considering the moderate number of T-cell epitopes obtained and the immune response generated, cell-mediated immunity was high and long lasting. The comparison made between predictive vaccine and S-2P mutant favours the interaction of B-cells with virus. Another important property of the vaccine was that it was less homologous to human proteins (17%), thus the chance of molecular mimicry and cross-reactivity was very low, providing the vaccine construct with no autoimmunity. The vaccine constructed was docked with immune receptors. The results indicate that the vaccine was interacting with TLR4 and TLR2, which induce an innate immune response. The vaccine was also interacting with B-cell receptors, which can elicit cellular or hormonal immune responses, while the interaction of the vaccine with the ACE2 receptor can lead to the blocking of the ACE2 receptor, which is responsible for the interaction and entry of the virus into the host (human). Further docking and experimental validation are required to confirm the above interactions and the overall effect of these interactions within the host.In the present study, the vaccine construct was based on spike proteins of wild-type Delta and Omicron. Several immuno-informatics tools are used to propose potentially antigenic and immunogenic epitopes, which will further help to design vaccines of the next generation. Omicron has the highest mutation in spike proteins, and the delta variant was, however, responsible for the second wave and high rate of effectiveness of COVID-19. The vaccine construct is based upon major SARS-Cov-2 variants to combat emerging variants like the XE virus, which is a combination of delta and omicron strains. The vaccine is constructed using 18-B-cell epitopes and 11-T-cell epitopes. Out of the total construct, ‘MSLGVENSV’ was found to be a novel epitope that may be used to generate neutralising antibodies for detection and neutralisation purposes. Although synthetically modified peptides have excellent prospects, as they have potential to improving stability and bioavailability. Experimental authentication is further required for validation of the outcome of the study. However, the vaccine was stable and found to interact with immune receptors and HLAs as well as with ACE-2 receptors, clearly indicating the potential for inducing humoral and cell-mediated immune responses. The C-ImmSim simulation observation strengthens the information like antibody release, cytokines release, and the state of B and T cells upon a single injection of vaccine as an antigen sufficient to provide immunity. Peptide-based therapeutics have various benefits over conventional molecules. Although repurposed medications as well as vaccines will serve as a topline of safeguard against COVID-19, future side effects of vaccination as well as resistance development as a consequence of drug administration also provide space for advancements in therapeutics.Additional file 1. Additional information supporting the study.


BackgroundCoronavirus disease 2019 (COVID-19) is widely recognized as a disease that affects the respiratory system, although it can also present with significant extrapulmonary symptoms. Very few studies have suggested an increased risk of gastrointestinal (GI) bleeding. This study aimed to elucidate the incidence, etiology, risk factors, and outcomes of clinically significant GI bleeding requiring endoscopic intervention in patients with COVID-19.MethodsThis is a case-control (1:2) retrospective analysis of all hospitalized adult patients with COVID-19 infection admitted between March 1, 2020, and January 5, 2021, in which we compared patients with upper and lower GI bleeds to those without. Cases are defined as patients hospitalized with COVID-19 who had a GI bleed requiring intervention while controls are defined as patients hospitalized with COVID-19 who did not have a GI bleed. Of 1002 patients admitted to the Albany Medical Center with COVID-19 infection, there were 76 confirmed cases of GI bleeding. These patients were compared to a control group composed of randomly selected patients with COVID-19 infection who were admitted to Albany Medical Center over the same time period. We assessed patients for in-hospital mortality, ventilator-free days on day 28, ICU-free days on day 28, and hospital-free days on day 28. Additional information collected included demographic information, comorbid conditions, COVID-19 treatments received, endoscopy findings, endoscopic treatment received, and if the patients required a packed red blood cell transfusion.ResultsOut of 1007 patients hospitalized with COVID-19, 76 (8%) had a GI bleed requiring endoscopic intervention. Peptic ulcer disease in the stomach or duodenum was the most common finding. The use of steroids, antiplatelet agents, and anticoagulation was not associated with an increased risk of GI bleed in COVID-19 patients. The GI bleed group required ICU care in 37% (28/76) compared with 21% (32/152) in the control group, which was statistically significant . Length of hospital stay was longer in the GI bleed group .ConclusionLength of hospital stay and ICU level of care was higher in the GI bleed group of patients with COVID-19. ICU level of care was noted to be associated with an increased risk of GI bleeding. A GI bleed in COVID-19 patients could be from the virus's direct effect on the gut mucosa or stress-induced bleeding like any other severely sick ICU patient; however, this needs to be explored in future studies. Severe acute respiratory syndrome coronavirus 2 (SARS-COV-2) is most commonly associated with respiratory illness . HoweverThis is a case-control (1:2) retrospective analysis of all hospitalized adult patients with COVID-19 infection who were admitted to Albany Medical Center between the dates of March 1, 2020, and January 5, 2021. This study was approved by the Albany Medical Center institutional review board and registered under protocol number 5825. Given the retrospective nature of our study, requirements for informed written consent were waived. COVID-19 was diagnosed by reverse transcription-polymerase chain reaction (RT-PCR) from a nasopharyngeal swab. GI bleeding was defined as overt upper and/or lower GI bleeding, including melena, hematochezia, hematemesis, or coffee-ground emesis, with a significant drop in hemoglobin of 2 g/dL or more from their baseline on admission with or without hemodynamic instability that required endoscopic intervention (i.e. upper endoscopy and/or colonoscopy).Inclusion criteria included adult patients 18 years of age and above who were admitted with COVID-19 and a clinically significant GI bleed as described above. The following COVID-19 patients were excluded: a) pregnant; b) incarcerated; c) patients with a GI bleed who did not receive endoscopic interventions.A total of 1007 patients admitted with a COVID-19 diagnosis during the study period were manually reviewed in our electronic medical record for a GI bleed according to our definition. In addition to GI consultation notes, an endoscopy database  was used to identify all patients during the study period who underwent endoscopy. After analyzing all data, 76 confirmed cases with a GI bleed requiring endoscopic intervention were identified. Our control group was selected randomly from the cohort of COVID-19-positive patients without a GI bleed in a 1:2 ratio using a random number generator.Patient data collected included: a) Demographics; b) Comorbidities; c) Laboratory parameters; d) COVID-19 treatment received: Remdesivir, anti-coagulation, corticosteroids; e) endoscopic findings; f) intervention done; clips, cautery, epinephrine injection or rectal packing; g) packed red blood cells transfusion; and h) outcome measured at day 28, including ventilator-free days, ICU-free days, and hospital-free days; and overall in-hospital mortality.We performed analyses to determine the incidence of gastrointestinal bleeds in hospitalized patients with COVID-19 infection. We also compared the clinical characteristics and outcomes of patients with COVID-19 infection who developed a significant gastrointestinal bleed requiring endoscopic intervention and the in-hospital mortality, ventilator-free days on day 28, ICU-free days on day 28, and hospital-free days on day 28 to the control group.Statistical analysisContinuous variables were represented by mean and standard deviation for normally distributed variables  and by the median and interquartile range (25th to 75th percentile) for non-normally distributed variables. Statistical inference for continuous variables was by the t-test for normally distributed variables and otherwise by the Mann-Whitney non-parametric test. Significance accepted at p<0.05. Categorical data were presented as frequencies and percentages with inference by Pearson’s chi-square test or Fisher’s exact test if the expected value in any cell was less than five.Kaplan-Meier survival curves were plotted to compare the in-hospital survival of COVID-19 patients with and without a GI bleed. The log-rank test was used to assess the difference between survival curves and the chi-square test was used to calculate the difference in survival between two groups at 120 days. Analysis was performed using Minitab  and R (v.3.6.1) statistical software (R Core Team (2020). R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria)  with a mean age of 67 (SD=16) years. The most common comorbidities noted were 54% (41/76) hypertension, followed by 33% (25/76) diabetes mellitus.A GI bleed was diagnosed at median day 9 IQR: 6-12) after hospitalization. In all cases, 100% of the patients (76/76) underwent esophagogastroduodenoscopy and 37% (28/76) underwent lower GI endoscopy in the form of either colonoscopy or flexible sigmoidoscopy. On upper endoscopy, peptic ulcer disease in the stomach or the duodenum was the most common finding in 47% (36/76), followed by gastritis and erosions in 9% (7/76). Variceal bleeding from gastroesophageal varices was noted only in one patient. On lower endoscopy, rectal ulcers were the most common finding in 53% (12/28). Endoscopic treatment was performed in 23 patients, with the use of endoclips being the most common intervention. Two patients required angiography and embolization, and four patients required rectal packing for bleeding rectal ulcers. Hemostasis was achieved in all cases, 84% (64/76) of the patients required an average of three units of packed red blood cell transfusion. The endoscopic findings and treatments are described in Table 2 after hWe randomly selected 152 controls as mentioned in the methods. The baseline characteristics were similar in the cases and controls as listed in Table The GI bleed group required ICU care in 37% (28/76) compared with 21% (32/152) in the control group, which was statistically significant . Length of hospital stay was longer in the GI bleed group . The 28-day all-cause mortality was not statistically significant between the two groups .In our analysis, 76 patients hospitalized with COVID-19 infection developed a GI bleed requiring endoscopic intervention. Our study found the incidence of GI bleeds in these patients was noted to be 8% as compared to previous studies showing a lower incidence of 1.5%-5.5% in the hospitalized patients in critical care units without COVID-19 infection . All patLimited literature has examined the incidence of GI bleeds in hospitalized patients with COVID-19 infection. The estimated incidence has been reported from 0.5% to 3%. Mauro et al. described the incidence of upper GI bleeds in the COVID-19 population in their multicentric study. The reported incidence of GI bleeds in their study was 0.47%, which was even lower than the reported rate of GI bleeds in non-COVID-19 patients. However, the generalizability of their findings is limited given that they excluded critically ill patients and patients with a lower GI bleed [The incidence of GI bleeds in non-COVID critically ill patients has been reported as anywhere from 1.5% to 5.5%, which is nearly similar to patients with COVID infection . The notThere are limited studies examining the risk factor for a GI bleed in hospitalized COVID-19 patients. The majority of these studies have shown no association between GI bleeds and the use of anticoagulation or steroids in this population, which aligns with our findings. However, one single-center study from Bronx Lebanon Hospital showed an increased risk of GI bleeds in COVID-19 patients with the use of steroids and anticoagulation. There are several limitations to their study, including selection bias, sample size, and definition for GI bleed . Few stuThere are several potential mechanisms of GI bleeds in hospitalized patients with COVID-19, which include the development of an inflammation-induced coagulopathy, direct damage of the virus on the gastrointestinal mucosa, stress ulcer from critical illness, and other factors, such as thromboprophylaxis, and steroids, which represent an additional risk factor for bleeding. At this point, it is not clear which is the dominant mechanism for the increased incidence of GI bleeds in COVID-19 patients. Marasco G et al. believe it may be because of critical illness and stress-induced ulcers, as reflected by a higher percentage of GI bleeding occurring in patients who are hospitalized with critical status in the ICU . In our To our knowledge, this is the first study aimed to study significant GI bleed in hospitalized patients with SARS-COV-2 infection requiring endoscopic intervention. Our study has several strengths, including a clear definition of GI bleeding in patients hospitalized with COVID-19 that required endoscopic intervention, a case-control study with a detailed comparison of clinical characteristics between the study group and the control group, and a relatively large number of the patient population who underwent endoscopic evaluation than previously reported. We also acknowledge some limitations in the study, including it being a retrospective, single-center study, a possible selection bias without randomization, and a lack of generalization to assess the risk of GI bleed in all hospitalized patients with COVID infection.In summary, we found that 8% of critically ill patients with COVID-19 infection developed a GI bleed requiring endoscopic intervention. Patients with longer ICU stays were likely to have a higher risk of GI bleed. Anticoagulation was not associated with an increased risk of a significant GI bleed in our study group. There was no statistically significant difference in the 28-day all-cause mortality between the two groups. Further large prospective studies are required to assess the risk and the pathophysiology of GI bleeding in hospitalized patients with COVID-19.


The expression of potential microRNA (miRNA) candidates isolated from serum and cerebrospinal fluid (CSF) exosomes of PD patients was assessed for their diagnostic value and their potential role as biomarkers for PD was explored. In this study, we characterize the expression level of miRNAs in the exosomes of blood sera and cerebrospinal fluid and explore their potential role as biomarkers for PD.  Parkinson's disease (PD) is a neurodegenerative disease, a hallmark by the formation of misfolded and aggregated  A total of 209 patients having an onset of PD, along with 60 neurodegenerative (ND) disorders and 50 healthy controls were enrolled. Blood samples and CSF samples were collected and exosomes were isolated. The isolated exosomes were characterized using CD63 detection and exosomal RNA was extracted. Serum miRNA profiling was carried out by synthesizing cDNA from the purified RNA and miRNA transcripts were determined by qRT-PCR using SYBR Green PremixScript. microRNA profiling strategy was employed for extracting the exosomal miRNAs from the exosomes.  Five common miRNAs viz. miR-151a-5p, miR-24, mir-485-5p, mir-331-5p, and mir-214 were found to be upregulated with statistical significance in both the serum exosome and CSF exosomes. The investigation revealed that serum and CSF exosomal miRNA molecules are definitive biomarkers for PD with proper specificity and sensitivity.  The significant level of miR-151a-5p, miR-24, mir-485-5p, mir-331-5p, and mir-214 was observed in the serum and CSF which may be established as a biomarker for the diagnosis of PD. The symJ-1, etc , 12. miRJ-1, etc . miRNAs J-1, etc . TherefoJ-1, etc . miRNA pJ-1, etc . BecauseAll experiments were carried out in accordance with the Declaration of Helsinki and approved by the Medical Ethics Committee of the Hospital . Written informed consent was obtained from all participants. The enrolled patients were free from severe medical conditions such as vascular diseases, hypertension, diabetes, and liver diseases.209 patients having an onset of PD were enrolled in the hospital between June 2018–July 2021 and were included in the study. All patients were investigated in the Neurology and Psychiatric Dept. of the Hospital. The PD patients were further evaluated for cognitive function and their behavioral and psychological symptoms were observed. Additional 60 neurodegenerative disorder (ND) patients (20 Alzheimer's disease (AD), 20 multiple sclerosis (MS), and 20 Huntington's disease (HD)) and another 50 healthy control participants were also enrolled from our institute who had no episodes of cognitive impairment or neurological disorder. The healthy control was also evaluated with similar cognitive examinations and brain CT scans. The baseline characteristics and demographic details of the participants were recorded and the onset of PD was characterized and evaluated.g for 20 min at 28°C. The serum samples were further preserved in a deep fridge (−80°C) for further analysis.Around 5 mL of blood samples were collected from 209 PD patients, 60 ND patients, and 50 healthy controls. Blood samples were collected through vein puncture using serum clot activator tubes. The blood samples were processed based on the standard operating procedure and specific guidelines of our institute. The blood samples were kept in a standing position for 15 min at 5°C and the serum was isolated by centrifugation at 3000 × g for 12 min. The CSF aliquots were then preserved at −80 C until further analysis. The samples were then analyzed for T-tau, Alpha-synuclein, and DJ-1 protein levels. The protein levels were determined and evaluated using a sandwich ELISA and Luminex assay.CSF samples were collected by puncturing the lumbar in polypropylene tubes and centrifuged at 1500 × g for 20 min followed by additional centrifugation at 7,500 × g for 8 min to remove the cell debris. The supernatants were then filtered using a filter membrane and around 500 μL of the debris was mixed with 0.3 volumes of the reagent and kept for incubation for 40 min at 6°C. The reagent mixed samples were further centrifuged at 9,000 × g for 12 min and the resultant pellets were treated with phosphate-buffered saline (PBS) solution. It was further centrifuged again, and re-suspended in prefiltrated PBS solution for the isolation of ribonucleic acid (RNA). Exosome preparations were also stored at −80°C for future use or until analysis.Exosomes were extracted from the serum and CSF samples using ExoQuick-TC  following the manufacturer's protocol. The samples were centrifuged at 2,000 × The isolated serum exosomes were characterized using a CD63 Detection reagent (Thermo) and further stained with a CD63 monoclonal antibody (Thermo) for the analysis of flow cytometry.CSF exosomes were characterized using the fluorescence-activated cell sorting(FACS) technique. The CSF samples were captured on Dynabeads and characterized using the CD63 monoclonal antibody. The beads were fluorescence-labeled with their respective anti-CD63, anti-CD86, and anti-CD54 controls and analyzed using BD FACS Calibur (BD Biosciences).g for 10 min at 4C for separating the upper aqueous phase. The resultant RNA pellets were then washed with a RPE buffer and eluted in nuclease-free water. The purified RNA was quantified using the Nanodrop ND-1000 (Thermo Fischer Scientific) and analyzed using real-time polymerase chain reaction (PCR) and quantitative polymerase chain reaction (qPCR).Exosomal RNA extraction was carried out from the supernatants using an EZ RNA Miniprep Kit  following the standard protocol. The samples were mixed properly and kept in a shaker for 2 min with uniform shaking and incubated at 34 C for 15 min. The samples were then centrifuged at 10000 × Serum miRNA profiling was carried out by synthesizing the complementary deoxyribonucleic acid(cDNA) from the purified RNA using a PrimeScript miRNA Standard Kit (United States) based on the standard protocol and cycling conditions. The reverse transcription was initiated by adding a poly-A tail to the 3' end and the process was followed by ligation with oligo-dT at the 5' end of the miRNA. CFS global miRNA profiling was carried out using TaqMan Low-Density Array Human miRNA Panels  by synthesizing the cDNA following the manufacturer's protocol and cycling conditions. The reverse transcription was initiated by quantitative PCR performed on an Applied Biosystems 7900HT thermocycler (Applied Biosystems). Furthermore, the exosome samples from the serum and CSF were analyzed using quantitative PCR and quantified by TaqMan miRNA assays  for the differential expression of miRNAs from the array panel.C(t) control as the reference value.The miRNA transcripts were determined by quantitative real-time PCR using the SYBR Green PremixScript miRNA real-time PCR kit (Applied Biosystem). The real-time PCR was carried out using an ABI thermocycler  using standard PCR conditions. All reactions were assayed and carried out in triplicate. The expression level of the miRNA was quantified by ΔCt by fixing at 0.05 unit of basal fluorescence and taking t-test was used for differentiating between the groups and P < 0.05 was taken for statistical significance. Receiver operation characteristic curves (ROC curve) were utilized to evaluate the sensitivity and specificity of miRNAs for possible diagnostic markers for Parkinson's disease.Statistical analysis was carried out using SPSS 17.0 . Student The demographic characteristics of the patients are presented in P < 0.05 compared to the healthy control. Whereas, in CSF samples, 19 exosomal miRNAs were upregulated and 8 miRNAs were underregulated with P < 0.05 compared to the healthy control (P < 0.05)), whereas the levels of miR-29b-2-5p and let-7d-5p were remarkably lower in the PD serum samples compared to healthy controls (P < 0.05). Similarly, the expression levels of miR-200a-5p, mir-22, mir-214, and mir-22 were significantly higher in CSF samples from patients with PD compared with the control group ((P < 0.05)) and the levels of mir-26a and mir-409-3p were significantly lower in CSF samples compared to controls (P < 0.05).CD63 protein is considered a common marker for exosomes. Furthermore, the serum and CSF-derived exosomes were verified for the presence of RNA. The exosomes were treated with RNAase solution mixed with 2% Triton and the degraded exosomal RNA was confirmed by running the gel using electrophoresis technique. The expression of exosomal miRNA in serum samples and CSF samples of PD patients were profiled based on TaqMan miRNA arrays, which comprise more than 900 miRNAs. The relative abundance of exosomal miRNAs was detected by normalizing the samples with the microRNA gene card RNU6-B. A total of 27 exosomal miRNAs showed differential expression in serum samples as well as in the CSF samples from PD patients. In the case of serum samples, 17 exosomal miRNAs were upregulated and 10 miRNAs were underregulated with  control . The expp-value and fold change compared to the healthy control and ND (Interestingly, miRNA viz. miR-151a-5p, miR-24, mir-485-5p, mir-331-5p, and mir-214 were expressed in both serum exosomes and CSF exosomes. These results suggest that these miRNAs may be the key miRNA for the early diagnosis of PD. These data revealed that miRNAs were present in serum and CSF exosomes. Moreover, it was confirmed that the exosomal miRNAs were differentially expressed in the serum and CSF samples of PD patients compared to healthy controls . For thel and ND . The heaThe Taqman assay observed that all miRNA viz miR-151a-5p, miR-24, mir-485-5p, mir-331-5p, and mir-214 were significantly overexpressed in the serum and the CSF exosome samples. Furthermore, the utility of serum miRNA and CSF miRNA was evaluated using ROC curves to distinguish their predictive abilities. The ROC results observed that the five common miRNAs from serum and CSF, which were differentially expressed, had a significant diagnostic ROC value for PD compared to healthy controls . The areα-syn. The progression of PD is not fully understood; however, the presence of α-syn is believed to play a crucial role in the disease progression. Exosomes are also thought to play a crucial role in the progression of PD [α-synuclein, tau protein, and DJ-1 in the CSF and brain tissue [The histopathological characterization of PD is mainly determined by the formation of the Lewy bodies and the presence of on of PD . On the on of PD . In thison of PD , 20 and on of PD , 22. Moron of PD , 24. Theon of PD . The stuon of PD . In our n tissue . FurtherThe use of RT-qPCR is regular and is widely for the analysis of miRNA because this technique is considered as a gold standard with high sensitivity and specificity . The RT-In conclusion, the detection of miR-151a-5p, miR-24, mir-485-5p, mir-331-5p, and mir-214 in the serum exosome and CSF exosomes may provide substantial aid in the early diagnosis of PD. However, other potent miRNAs may also establish a significant relationship with PD and this could not be observed in the present study because of the limited sample size. Therefore, the present study concludes that the significant level of miR-151a-5p, miR-24, mir-485-5p, mir-331-5p, and mir-214 in the serum and CSF may be established as a biomarker for the diagnosis of PD.